Abstract
In this work we analyze splicing mutations identified in propionic acidemia patients to clarify their functional effects and their involvement in the disease phenotype. Two mutations in the PCCA gene detected in homozygous patients and involving consensus splice sequences (IVS21+3del4 and IVS22-2A>G) were shown to produce some normal splicing in patients’ cells, at very low levels, which were quantitated by real-time PCR methods, and which presumably are sufficient to moderate the phenotype. We have also analysed the effect of mutations c.653A>G and IVS10-11del6 in the PCCB gene present in heterozygous patients with mild phenotype. The c.653A>G mutation is located in the last codon of exon 6 and interferes with the correct spliceosomal assembly activating a cryptic splice site within exon 6, which leads to an in-frame six-nucleotide deletion (delV217-K218). Minigene analysis and sequence-specific hybridization probes using real-time PCR methods showed that no normally spliced transcript is detectable in the patients’ fibroblasts. The IVS10-11del6 mutation shortens the polypyrimidine tract of the 3′-splice site of exon 11, resulting in exon skipping. Some normal transcript is detectable by allele-specific hybridization probes. These analyses suggest that, in some cases, the regulation of gene splicing can potentially play an important role in human disease influencing phenotypic parameters.
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Acknowledgements
The authors wish to thank Dr. Andresen for his advice on minigenes and A. Sánchez, F. Leal and R. Navarrete for technical assistance. This work was supported by grants from Fondo de Investigaciones Sanitarias (01/0405, REDEMETH G03/054 and RECGEN C03/07). S.C. is supported by Comunidad Autónoma de Madrid. The financial support of Fundación Ramón Areces to the Centro de Biologia Molecular “Severo Ochoa” is gratefully acknowledged.
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Clavero, S., Pérez, B., Rincón, A. et al. Qualitative and quantitative analysis of the effect of splicing mutations in propionic acidemia underlying non-severe phenotypes. Hum Genet 115, 239–247 (2004). https://doi.org/10.1007/s00439-004-1147-1
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DOI: https://doi.org/10.1007/s00439-004-1147-1